Immunogenic compositions against gastrin peptides

ABSTRACT

Immunogenic compositions useful for the treatment of ulcers or tumors whose growth is dependent on or stimulated by gastrin hormones are disclosed. The immunogenic compositions induce antibodies in a subject which selectively neutralize the specific hormones. Pharmaceutical compositions comprising effective amounts of the immunogenic compositions and methods of treatment using the compositions are disclosed. A method of reversing the inventive treatments by neutralizing the antibodies induced in vivo is also disclosed.

This application is a continuation-in-part of application Ser. No.07/351,193, filed May 12, 1989, now abandoned, which is acontinuation-in-part of application Ser. No. 07/301,353, filed Jan. 24,1989 and issued Jun. 11, 1991 as U.S. Pat. No. 5,023,077.

BACKGROUND OF THE INVENTION

Peptic ulcer disease exists in two forms, duodenal ulcers and gastriculcers. Central to the cause of duodenal ulcers, is the production ofexcess stomach acid and pepsin and a rapid gastric emptying time. Thisresults in an increase in duodenal exposure to secreted acid andenzymes, and in mucosal damage.

The second form of the disorder, gastric ulcer disease, may be caused byincreased stomach acid and a breakdown of the complex stomach defensesthat normally protect the gastric mucosa from acid damage. Although thetwo conditions have different etiologies, both benefit from a reductionin gastric acid secretion.

Because excess stomach acid is a central cause of ulcers, antacidpreparations are commonly used as one method of treatment. This methodmerely neutralizes stomach acid after it is produced. Consequently,large quantities of antacids must be consumed on an ongoing basis toneutralize acid which is continually produced in the stomach. Antacidsdo not cure the disease because they do not affect the source of acidproduction.

Gastric acid is produced in a specialized stomach cell, the parietalcell. Parietal cells can be stimulated to secrete acid by acetylcholine,histamine and gastrin, upon the binding of each of these compounds withspecific receptors on the surface of the cell. Of these the most potentstimulator of acid secretion is the peptide hormone gastrin.

Current approaches to the control and cure of peptic ulcers center upondevising drugs that inhibit the ability of one or more of thesecompounds to stimulate acid production or secretion. The most effectivegroup of drugs approved for sale are the H2 antagonists (e.g. Tagamet®and Zantac®) which block the histamine H2 receptors on gastric parietalcells and inhibit acid secretion. These drugs, however, requirerelatively large doses on a daily basis and may induce severalundesirable side effects. In cases where H2 antagonists have curedulcers, relapses occur in almost 100% of cured individuals within a yearof discontinuation of treatment. Other drugs have also exhibitedproblems, including low efficacy and unacceptable levels of toxicity. Inthe case the peptide hormone gastrin, no successful chemical antagonistshave been identified.

Gastrin has several important functions in the gastrointestinal tract,the two most important being stimulation of acid secretion andstimulation of the growth of cells in the gastrointestinal tract. Thehormone exists in at least two molecular forms, heptadecagastrin ("G₁₇") and tetrateseracontagastrin ("G₃₄ ") named according to the number ofamino acid ("AA") residues in each molecule. G₃₄ and G₁₇ are identicalin structure at the carboxy terminus, which is the binding site of thehormones with receptors. G₁₇ constitutes the 17 carboxy terminal("C-terminal") end residues of G₃₄. G₃₄ consists of the 17 C-Terminalend residues which comprise G₁₇ and an additional different amino acidsequence of 17 amino terminal ("N-terminal") residues. When G₃₄ is splitby trypsin a G₁₇ subunit and a non-hormonal 17 amino acid subunitresults. Though G₁₇ is usually obtained by trypsin cleavage of G₃₄, eachform may also be generated separately from its own prohormone.

Although G₁₇ and G₃₄ are thought to be equipotent on a molar basis asstimulator of acid release, G₃₄ is most probably responsible for thestimulation of growth of the gastrointestinal mucosa and the maintenanceof the basal acidity of the stomach. G₃₄ is the principal form presentduring interdigestive periods. G₃₄ has a serum half life approximatelysix times as long as G₁₇ (40 minutes versus 6 minutes) and is producedin both the stomach and the duodenum. Alternatively, G₁₇ is the primarystimulator of meal-induced gastric acid secretion. G₁₇ is 1500 timesmore potent than histamine and makes up 90% of the antral (stomach)gastrin. G₁₇ accounts for roughly 60%-70% of the gastrin-mediated acidrelease.

The prior art in the area of gastrin immunology mainly concerns theinduction of antibodies useful for identifying anatomic sites containingor producing gastrin G₁₇ or G₃₄ in laboratory animals; see Sugano, K.,et al., 1985, "Identification and characterization of glycine-extendedpost translational processing intermediates of progastrin in porcinestomach", J. of Biological Chemistry 250: 11724-11729; Vaillant, C., etal., 1979, "Cellular origins of different forms of gastrin: The specificimmunocytochemical localization of related peptides. J. HistochemCytochem 27:932-935; Larsson, L. I. et al., 1977, "Characterization ofantral gastrin cells with region-specific antisera". J. Histochem.Cytochem 25: 1317-1321. The antisera reported in these publicationscontained antibodies of numerous specificities, for a variety ofantigenic epitopes on gastrin molecules.

Attempts to control gastrin levels by anti-gastrin antibodies induced byactive immunization or passive administration of preformed antibodiessuch as those reported in Jaffe, B. M., et al., 1971, "Gastrinresistance following immunizations to the C-terminal tetrapeptide amideof gastrin, Surgery 69: 232-238; Jaffe, B. M., et al., 1970, "Inhibitionof endogenous gastrin activity by antibodies to the carboxyl terminaltetrapeptide amide of gastrin", Gastroenterology 58: 151-156; Jaffe etal., 1969, "Inhibition of endogenous gastrin activity by incubation withantibodies to the C-terminal tetrapeptide of gastrin. Surgery 65:5633-639 are different from the present invention in that the immunogenused was derived from the carboxyl terminal tetra-peptide amino acidsequence common to G₁₇, G₃₄, and to another important hormone,cholecystokinin ("CCK"). The immunogen of Jaffe et al. is thus of nopractical value as an anti-gastrin vaccine component; on the contrary,it would produce a deleterious state in which all gastrin activity andother hormone function of G₁₇, G₃₄, together with CCK, would be blockedand eliminated by immunization.

This invention provides a novel immunological approach to the controland regulation of gastrin induced disorders such as peptic ulcers.According to the invention, antibodies are induced in the patient byactive immunization with immunogens that selectively target specificforms of gastrin. Alternatively, the patient can be passively immunizedwith anti-gastrin antibodies specific for certain forms of gastrin.

In addition to peptic ulcers, other diseases appear to be related to thehormonal and stimulatory effects of gastrin. These diseases may also betreated by the selective anti-gastrin treatment of the invention.

An area of major medical importance for which the neutralization ofgastrin hormonal activity has great therapeutic potential concerns thecontrol of tumors and pathological conditions that are stimulated bygastrointestinal hormones. Several cancers of the gastrointestinal tractand associated tissues are stimulated to grow by the trophic action ofgastrin. See Lamers, C.S.H.S., and Jansen, J.B.M.S., 1988, "Role ofGastrin and Cholecystokinin in Tumours of the Gastrointestinal Tract",Eur. J. Cancer Clin. Oncol. 22: 267-273. Gastrin promotes the growth ofcolon carcinoma, gastric carcinoma and gastric carcinoids. Gastrinantagonists may inhibit the growth of human colon cancer and enhancehost survival as has been shown in mice; see, Beauchamp, R. O., et. al.1985, "Proglumide, A Gastrin Receptor Antagonists, Inhibits Growth OfColon Cancer And Enhances Survival In Mice." Ann. Surg. 202: 303-309.The neutralization of gastrin tumor promoting activity may provide animportant therapy for these diseases.

A second important application of gastrin neutralization therapyconcerns conditions in which the hormone is overproduced. Certaincancers of the gastrointestinal tract, apudomas, produce extremely largequantities of gastrin. In either case, the excess hormone produced bythe apudoma or pituitary tumor will have adverse physiologic effects onorgans or tissues containing receptors for the hormone. Excess gastrinproduction by apudomas stimulates hypertrophy of the acid secretingepithelium of the stomach, leading to excess stomach acid secretion,peptic ulcer, and neoplastic changes in the epithelium.

Available treatment for tumors stimulated by gastrin and for tumors thatproduce gastrin consists primarily of surgical resection of thecancerous tissue. This approach is frequently unsuccessful; in manyinstances the tumors cannot be located or are present in anatomic sitesthat are inoperable. In most instances these tumors do not respond wellto radiation or chemotherapy regimens. New treatments are needed tosupplement present procedures.

A therapeutic method of selectively neutralizing the biological activityof these hormones would provide an effective means of controlling orpreventing the pathologic changes resulting from excessive hormoneproduction.

The method of cancer therapy described in this invention has severaladvantages over present treatment methods. The method is non-invasive,selectively reversible, does not damage normal tissue, does not requirefrequent repeated treatments, does not cross the blood brain barrier andhas reduced side effects.

The therapy may be selectively reversed by injecting the patient with apharmaceutical composition comprising a neutralizing epitope molecule.This molecule should comprise the epitope sequence free of animmunogenic carrier. This non-immunogenic molecule will bind to the freeantibodies previously induced against the epitope in the host.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: illustrates the stomach acid secretions over time of a controlrat injected sequentially with hG₁₇, antisera raised against anunrelated peptide and hG₁₇.

FIG. 2: illustrates the stomach acid secretion over time or a ratinjected sequentially with hG₁₇, hG₁₇ premixed with anti hG₁₇ antisera;and hG₁₇.

FIG. 3: illustrates the stomach acid output over time in a rat activelyimmunized against G₁₇ in response to an injection of pentagastrin ("pG")followed by injections of G₁₇.

FIG. 4: illustrates the stomach acid output over time in a rat activelyimmunized against G₁₇ in response to an injection of G₁₇ followed byinjections of pentagastrin and G₁₇.

FIG. 5: illustrates the stomach acid output over time in a control ratin response to the sequential injection of pG, G₁₇ and pG.

FIG. 6: illustrates stomach acid output over time in a rat activelyimmunized against G₁₇ in response to sequential injections of G₃₄, G₁₇and G₃₄.

FIG. 7: illustrates stomach acid output over time in a control rat inresponse to sequential injections of G₃₄, G₁₇ and G₃₄.

FIG. 8: depicts the binding capacity in picograms ("pg.") of Antigen permicroliter ("ul") of sera of anti-G₁₇ antibodies induced by twosynthetic peptide antigen epitope polymers of the invention.

FIG. 9: illustrates the total quantity of stomach acid secreted by hG₁₇-immune rats and non-immune rats in response to graded doses of hG₁₇.Two non-immune rats and four hG₁₇ -immune rats were injected with 0.12micrograms ("ug") and 2.5 ug doses of hG17. Two hG₁₇ -immune rats wereinjected with doses of 25 ug and 250 ug of hG₁₇.

FIG. 10: depicts the stimulation of the growth of human colon cancercell line, HCT 116, by pentagastrin.

FIG. 11: illustrates the effect of anti-G₁₇ antibodies on the growth ofcolon cancer implants in nude mice infused with G₁₇, as measured by meantumor volume in cubic millimeters. Group I consisted of rats injectedwith rat anti-G₁₇ antibodies, and Group II consisted of rats injectedwith normal rat antibodies.

FIG. 12: illustrates the effect of anti-G₁₇ on the growth of coloncancer implants in nude mice as measured by mean tumor volumes in cubicmillimeters. Group 1 was injected with both G₁₇ and anti-G₁₇ antibodies.Group III was injected only with saline.

DESCRIPTION OF THE INVENTION

Since the different forms of gastrin vary in function, it is necessaryto selectively neutralize specific forms of gastrin to control specificfunctions. To regulate gastrin-mediated secretion of stomach acidfollowing meals (the principal source of excess stomach acid relating toulcers), an immunogen must specifically target G₁₇. In order toselectively neutralize G₁₇, one or more antigenic epitopes on G₁₇ thatare not found on G₃₄ or cholecystokinin which exhibits carboxy terminalhomology with gastrin must be identified. As discussed above even thoughthe C-terminus of G₁₇ and G₃₄ are identical the N-terminus of G₁₇ isvery different from that of G₃₄. This results in antigenic epitopes thatare unique to G₁₇ and can be separately targeted. We have identified andmapped such a unique epitope on G₁₇. A specific embodiment of thepresent invention concerns immunogens comprising this unique epitope.These immunogens result in high levels of anti-G₁₇ antibodies that donot crossreact with G₃₄ and block some or all of G₁₇ stimulation ofgastric acid secretion while still allowing G₃₄ and CCK, which sharewith G₁₇ a common receptor, to carry out their physiologic function. Theregulation of acid secretions can also involve the neutralization of G₃₄; we have also identified and mapped unique epitopes on G₃₄ that are notfound on G₁₇ or CCK.

Our immunoneutralizing approach has several attractive advantages overcurrent treatments for peptic ulcer. One of these advantages is theovercoming of the major problem of patient compliance since a daily doseof a drug is not required. This invention treats ulcers by preventingthe release of excess stomach acid, unlike antacids that neutralizesecreted acid. By administering our synthetic peptide as an immunogen,the frequency and quantity of treatment administration is decreased,while at the same time long-lasting control of acid production, longterm prevention of recurrence, and reduced side effects and easierpatient administration are provided. Unlike conventional anti-ulcerdrugs, antibodies generated by the peptide immunogens are very specificto their target. They do not cross the blood-brain barrier, and theiruse avoids certain complications encountered with drugs, for example,liver toxicities associated with H₂ antagonists. In addition, unlikethis invention, agonists or antagonists of G₁₇ have reduced efficencyfor controlling ulcers because such compounds have low specificity forthe receptors for G₃₄ and CCK, which have identical receptor bindingsites with G₁₇.

The immunogens against one form of gastrin, "little gastrin", or G₁₇,are constructed to produce an anti-gastrin immunogen component that willinduce a selective and specific antibody response to G₁₇ in theimmunized human or other vertebrate, but not to G₃₄ or CCK. Thisselective immunization to produce G₁₇ specific antibodies is crucial toavoid producing antibodies specific for or cross reactive with G₃₄,which might during the treatment of a specific condition induceundesirable side effects by blocking G₃₄ physiologic functions. Theantibodies resulting from the immunization with such immunogens targetthe chemical structure of G₁₇ which is antigenically and immunogenicallyunique from the structure of G₃₄.

Peptides comprising the amino acid residues beginning from the aminoterminus (amino acid residue number one) of G₁₇ and extending up to andincluding amino acid residue number 12 having the sequencepyro-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr, are used toprepare the immunogens of the invention by coupling them to animmunogenic carrier. For simplicity this sequence can be written basedupon the international code for amino acids aspyro-E-G-P-W-L-E-E-E-E-E-A-Y. The immunogens may contain a part or allof this sequence. The last 5 carboxy-terminal end amino acids of the G₁₇chemical structure (residues 13-17) are preferably not used, becausethis sequence is a common antigenic sequence between G₁₇, G₃₄, and atleast one other hormone, cholecystokinin (CCK). Fragments, extensions,or other subsets of the natural hormone and of this 12 amino acidsequence of G₁₇ may be used.

The peptides which may be used to prepare the immunogens of theinvention may comprise one of the following amino acid sequences:pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-,pGlu-Gly-Pro-Trp-Leu-Glu-, pGlu-pGly-Pro-Trp-Leu-Glu-Glu-,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-, pGlu-Gly-Pro-Trp-Leu-,PGlu-Gly-Pro-Trp-, pGlu-pGly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu.

The most preferred peptide used in the invention is the hexamerpGlu-Gly-Pro-Trp-Leu-Glu-. The other peptides such aspGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-, pGlu-Gly-Pro-Trp-Leu-andpGlu-Gly-Pro-Trp are also preferred. One or more other amino acids mayalso be substituted for those of the natural sequence, so that increasedor decreased binding capacity, specificity and/or titer of the antibodyresponse against G₁₇ may be induced in the vaccinated host by theimmunogen.

In other embodiments of the invention the use of preformed G₁₇ specificpolyclonal and/or monoclonal antibodies and their derivatives orfragments produced by immunization, hybridoma, recombinant DNA or othertechnologies as a method of passive immunization for the control ofgastric acid secretion stimulated by G₁₇ may be used.

The present invention also provides immunogens against a second form ofgastrin, "big gastrin" or G₃₄. These immunogens are used to produce ananti-gastrin vaccine component that may be useful for the treatment orprevention of other gastrointestinal diseases and that will induce aselective and specific antibody response to G₃₄ (but not to G₁₇ or CCK)in the vaccinated human or other vertebrate. This selective immunizationto produce G₃₄ specific antibodies is crucial to avoid producingantibodies specific for or cross reactive with G₁₇.

The G₃₄ immunogens specifically target chemical structures of G₃₄ whichare antigenically and immunogenically unique from the structure of G₁₇.The chemical structures of G₃₄ utilized in this invention include, butare not limited to, peptides comprising the amino acid residuesbeginning from the amino terminus (amino acid residue number one) of G₃₄and extending up to and including amino acid residue number 22. Thesequence of this peptide ispyro-Glu-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His-Leu-Val-Ala-Asp-Pro-Ser-Lys-Lys-Gln-Gly-Pro-Trp-Leu.Based upon the international code for amino acids, this sequence ispyro-E-L-G-P-Q-G-P-P-H-L-V-A-D-P-S-K-K-Q-G-P-W-L-. The G₃₄ immunogensmay contain part or all of this sequence and comprise the sequencecoupled to an immunogenic carrier. The sequence of the last 12 aminoacids of the G₃₄ chemical structure (residues 23-34) are preferably notused in this invention because this sequence is a common antigenicsequence between G₁₇ and G₃₄. The sequence of amino acids are also notused since the sequence 29-34 has common antigenic sites withcholecystokinin. It is contemplated that the use of any fragments,extensions, or other subsets of the natural hormone and of this 22 aminoacid sequence may be used as immunogens.

The most preferred peptide for use is the hexamerpGlu-Leu-Gly-Pro-Gln-Gly. The peptidespGlu-Leu-Gly-Pro-Gln-Gly-Pro-Pro-His- andpGlu-Leu-Gly-Pro-Gln-Gly-Arg-Pro-Pro-Pro-Pro-Cys are also preferred. Oneor more other amino acids may also be substituted and/or modified forthose of the usual natural sequence, so that increased or decreasedbinding capacity, specificity and/or titer of the antibody responseagainst G₃₄ may be induced in the vaccinated host by the immunogen.

Preformed G₃₄ specific monoclonal antibodies and their derivatives orfragments produced by hybridoma, recombinant DNA or other technologiesmay also be used as a method of passive immunization for the control ofgastric acid secretion stimulated by G₃₄.

The peptides and immunogens may be produced by any process commonly usedin the art including, for example, standard peptide synthesistechnologies; methods employing recombinant DNA and associatedtechnologies; antigen mimicking methods including antibody-internalimage technology and any other related methodologies that produce astructure that immunologically resembles the antigenic structures of(mimotopes).

The means by which anti-gastrin antibodies prevent acid release has notbeen thoroughly established. Without being bound by theory, we believethat the acid suppressive effect of our immunogen is due to the bindingof anti-gastrin antibodies to gastrin (G₁₇ and/or G₃₄) in the blood, andthereby preventing the binding of gastrin to its physiological receptorson the surfaces of parietal cells. Thus, gastrin is prevented fromsignaling parietal cells to secrete acid into the stomach.

For the G₁₇ and/or the G₃₄ epitopes of this invention to induceantibodies, it may be necessary to increase their immunogenicity bychemically coupling them to other molecules. Such molecules are termed"carriers". Any molecule capable of serving as a carrier may be used.Examples of carriers for this purpose include: diphtheria toxoid,tetanus toxoid, keyhole limpet hemocyanin, bovine serum albumin, etc.Fragments of these carriers, including single epitopes, may also beused. Any method of chemically coupling the epitopes to the carriers maybe followed. A preferred method utilizes the bifunctional linking agentEMCS described in U.S. Pat. No. 4,302,386; Lee et al., 1981.

The epitopes can be alternatively rendered immunogenic by crosslinking(e.g., polymerizing) a number of epitopes. For this purpose, it may benecessary to extend the molecule bearing the G₁₇ or G₃₄ epitope by theaddition of selected compounds that provide structures through which thecrosslinking will occur. These additions must not disrupt the structureof the gastrin epitope, because the capacity to induce anti-gastrinantibodies would be lost. For example, to the carboxy terminal end ofthe G₁₇ epitope pyro-E-G-P-W-M-E-E is added the amino acid sequenceK-R-P-P-P-P-K, to give pyro-E-G-P-W-M-E-E-K-R-P-P-P-P-K. This moleculeis then polymerized with glutaraldehyde, which crosslinks thelysine(K)residues, to form the crosslinked immunogen. This crosslinkedimmunogen should induce specific antibodies against G₁₇.

It may be desirable in some applications to immunize against both G₁₇and G₃₄. In this embodiment G₁₇ and G₃₄ immunogens are used incombination including optionally an immunogen with an epitope common toG₁₇ and G₃₄, e.g. Asp-Pro-Ser-Lys-Lys-Glu-Pro-Trp-Leu-, so thatantibodies against both G₁₇ and G₃₄ are induced by the immunized host.The immunogens of this invention are therefore useful for more than justthe treatment or prevention of ulcers. The immunogens may be used totreat any disease in which the gastrin stimulated secretion of stomachacid or stimulation of the growth of cancer by gastrin (e.g. colorectaland gastric) is a factor.

Administration of these immunogens, compositions containing them, orpharmaceutically acceptable and immunologically effective derivativesthereof, may be via any of the conventionally accepted modes ofadministration of agents which exhibit immunogenicity.

The compositions in which the immunogens are administered may be in avariety of forms. These include, for example, solid, semi-solid andliquid dosage forms, such as powders, liquid solutions or suspensions,suppositories, and injectable and infusible solutions. The preferredform depends on the intended mode of administration and therapeuticapplications. The compositions also will preferably include conventionalpharmaceutically acceptable carriers and may include other medicinalagents, carriers, adjuvants, excipients, etc., e.g., human serum albuminor plasma preparations. Preferably, the compositions of the inventionare in the form of a unit dose. The amount of active compoundadministered as an immunization or as a medicament at one time, or overa period of time, will depend on the subject being treated, the mannerand form of administration, and the judgment of the treating physician.However, an effective dose may be in the range of from about 1 ug toabout 10 mg of the immunogen of this invention, preferably about 100 ugto about 2 mg; it being recognized that lower and higher doses may alsobe useful.

This invention provides a novel immunological approach to the treatmentof tumors whose growth is dependent upon or stimulated by one or more ofthe forms of gastrin. According to the invention, antibodies are inducedin the patient by passive or active immunization with immunogens thattarget one or more of the forms of gastrin. Such antibodies will bind toand neutralize the hormone to which the antibodies are directed, therebypreventing the hormone from acting upon the tumor. The antibody-mediateddeprivation of hormonal activity thus constitutes a means of controllinggastrin dependent tumors.

This invention additionally provides a means of treatment for theeffects of tumors that produce one or more forms of gastrin. In thisembodiment, the immunogens of this invention are used to induceantibodies against the form of the hormone that is produced by thetumor. Such antibodies will bind to and neutralize the hormone, therebypreventing pathological consequences resulting from abnormally elevatedlevels of hormone.

This invention also provides a method for selectively reversing theantibody-mediated immunity induced by the anti-hormone immunogens of theinvention. Soluble monovalent, monodeterminant epitopes are injectedinto the patient to bind to and neutralize the selected anti-hormoneantibodies. The antibodies will then be incapable of binding additionalquantities of hormone, and will no longer affect the hormone'sbiological activity.

The immunogens of this invention can be constructed to induce antibodiesthat specifically neutralize a single form of gastrin. This isadvantageous in treating a tumor and pathologic condition, particularlyone that is stimulated primarily or totally by a single form of gastrin.Alternatively, these specific immunogens can be selectively combined toproduce an immunogen, in which the immunogen's individual constituentseach induce antibodies that target distinct forms of gastrin. Combinedimmunogens would be used to treat tumors and conditions that arestimulated by more than one form of gastrin. Such an immunogen has theadvantage of allowing for the addition or removal of individualimmunogen constituents. The specificity of the antibody response canthus be controlled over time, thus enabling the physician to tailor theongoing treatment to the needs of individual patients.

This example demonstrates a means of preparing immunogens to induceanti-G₄₇ or anti-G₃₄ antibody responses.

EXAMPLE 1

Peptides for the induction of specific immune responses to either G₁₇ orto G₃₄ were prepared by standard solid state synthesis methods. Eachpeptide was characterized as to amino acid content and purity.

Peptides with the following amino acid sequences were synthesized:

    ______________________________________                                        Peptide 1-                                                                            Human G.sub.17 (1-6) ("hG.sub.17 (6)"): pGlu--Gly--Pro--                      Trp--Leu--Glu--Arg--Pro--Pro--Pro--Pro--Cys                           Peptide 2-                                                                            Human G.sub.17 (1-5) ("hG.sub.17 (5)"): pGlu--Gly--Pro--                      Trp--Leu--Arg--Pro--Pro--Pro--Pro--Cys                                Peptide 3-                                                                            Human G.sub.17 (1-4) ("hG.sub.17 (4)"): pGlu--Gly--Pro--                      Trp--Arg--Pro--Pro--Pro--Pro--Cys                                     Peptide 4-                                                                            Rat G.sub.17 (1-6) Leu 5 ("rG.sub.17 (6)Leu 5"): pGlu--Arg--                  Pro--Pro--Leu--Glu--Arg--Pro--Pro--Pro--Pro--                                 Cys                                                                   Peptide 5-                                                                            Human G.sub.34 (1-6) ("hG.sub.34 (6)"): pGlu--Leu--Gly--                      Pro--Gln--Gly--Arg--Pro--Pro--Pro--Pro--Cys                           Peptide 6-                                                                            Human G.sub.34 (13-22) ("hG.sub.34 /G.sub.17 combination"):                   Asp--                                                                         Pro--Ser--Lys--Lys--Gln--Gly--Pro--Trp--Leu--                                 Pro--Pro--Pro--Pro--Cys                                               ______________________________________                                    

Each of these peptides were conjugated to amino groups present on acarrier such as Diphtheria toxoid ("DT") via the terminal peptidecysteine residue utilizing hetero-bifunctional linking agents containinga succinimidyl ester at one end and maleimide at the other end of thelinking agent.

To accomplish the linkage between any of Peptides 1-6 above and thecarrier, the dry peptide was dissolved in 0.1M Sodium Phosphate Buffer,pH 8.0, with a thirty molar excess of dithiothreitol ("DTT"). Thesolution was stirred under a water saturated nitrogen gas atmosphere forfour hours. The peptide containing reduced cysteine was separated fromthe other components by chromatography over a G10 Sephadex columnequilibrated with 0.2M Acetic acid. The peptide was lyophilized andstored under vacuum until used. The carrier was activated by treatmentwith the hetero-bifunctional linking agent e.g. Epsilon-maleimidocaproicacid N-hydroxysuccinimide ester, ("EMCS"), in proportions sufficient toachieve activation of approximately 25 free amino groups per 10⁵molecular weight of carrier. In the specific instance of diphtheriatoxoid, this amounted to the addition of 6.18 mg of EMCS (purity 75%) toeach 20 mg of diphtheria toxoid.

Activation of diphtheria toxoid was accomplished by dissolving each 20mg aliquot of diphtheria toxoid in 1 ml of 0.2M Sodium Phosphate Buffer,pH 6.45. Aliquots of 6.18 mg EMCS were dissolved into 0.2 ml of DimethylFormamide ("DMF"). Under darkened conditions, the EMCS was addeddropwise in 50 microliter ("ul") amounts to the DT with stirring. After2 hours of incubation in darkness, the mixture was chromatographed on aG50 Sephadex column equilibrated with 0.1M Sodium Citrate buffer, pH6.0, containing 0.1 mM EDTA.

Fractions containing the EMCS activated diphtheria toxoid wereconcentrated over a PM 10 ultrafiltration membrane under conditions ofdarkness. The protein content of the concentrate was determined byeither the Lowry or Bradford methods. The EMCS content of the carrierwas determined by incubation of the activated carrier with cysteine-HClfollowed by reaction with 10 mM of Elman's Reagent 5,5'dithio-bis(2-nitrobenzoic acid) 10 mM. The optical density difference between ablank tube containing cysteine-HCl and the sample tube containingcysteine-HCl and carrier was translated into EMCS group content by usingthe molar extinction coefficient of 13.6×10³ for 5-thio-2-nitro benzoicacid at 412 nm.

The reduced cysteine content (--SH) of the peptide was also determinedutilizing Elman's Reagent. Approximately 1 mg of peptide was dissolvedin 1 ml of nitrogen gas-saturated water and a 0.1 ml aliquot of thissolution was reacted with Elman's Reagent. Utilizing the molarextinction coefficient of 5-thio-2-nitro-benzoic acid (13.6×10³), thefree cysteine --SH was calculated. An amount of peptide containingsufficient free --SH to react with each of the 25 EMCS activated aminogroups on the carrier was dissolved in 0.1M Sodium Citrate Buffer, pH6.0, containing 0.1 mM EDTA, and added dropwise to the EMCS activatedcarrier under darkened conditions. After all the peptide solution hadbeen added to the carrier, the mixture was incubated overnight in thedark under a water saturated nitrogen gas atmosphere.

The conjugate of the peptide linked to the carrier via EMCS is separatedfrom other components of the mixture by chromatography over a G50Sephadex column equilibrated with 0.2M Ammonium Bicarbonate. Theconjugate eluted in the column void volume is lyophilized and storeddesiccated at -20° C. until used.

The conjugate may be characterized as to peptide content by a number ofmethods known to those skilled in the art including weight gain, aminoacid analysis, etc. Conjugates of Peptides 1-6 and diphtheria toxoidproduced by these methods were determined to have 20-25 moles of peptideper 10⁵ MW of carrier and all were considered suitable as immunogens forimmunization of test animals.

EXAMPLE 2

As examples of the utilization of peptides containing sequences of humangastrin as immunogens to induce immune responses against hG₁₇ or hG₃₄,we have immunized rats with the conjugate immunogens constructed fromPeptides 1-6 of Example 1 and Diphtheria toxoid ("DT") (referred to asImmunogens 1-6, respectively).

Six different groups of 15 Sprague-Dawley female rats (200 gm. bodyweight) were each immunized with one of the immunogens constructed fromPeptides 1-6. Each animal was injected subcutaneously with 0.25 ml ofimmunogen consisting of 0.1 mg of conjugate dissolved in 0.125 ml of0.1M Sodium Phosphate Buffered Saline, pH 7.3, emulsified with an equalvolume of Squalene-Arlacel (4:1 ratio volume/volume) vehicle containing0.05 mg of Nor MDP as adjuvant.

Two additional groups of 15 rats were immunized with a peptide-DTconjugate in which the peptide had no sequence homology with gastrins soas to act as a negative immunization control.

Each rat was given an injection of immunogen at 0, 3, and 6 weeks. Bloodwas collected from each rat at 3, 6, and 8 weeks of the experiment.Serum was collected from each blood sample and stored at -20° C. untilutilized in assays to determine the presence of anti-gastrin antibodies.

Two types of assays were used to detect anti-gastrin antibodies. Asolid-phase enzyme linked immunosorbent assay (ELISA) and a liquid phaseradioimmunoassay (RIA) were employed.

ELISA was used to screen for reaction or cross reaction of antiseraraised against Peptides 1-6 with Peptides 1-6 or with hG₁₇, hG₃₄, orhCCK. The RIA was used to quantitate the antibody levels in theantiserum of each immunized animal that was reacted with hG₁₇ or hG₃₄ bydetermining the antigen binding capacity (ABC), expressed as pg hormonebound per ("ul") of antiserum (pg/ul).

The ELISA was conducted by coating polystyrene 96 well plates (ImmulonII) with 1 ug/ml of Peptides 1-6, hG₁₇, hG34, or hCCK antigen. Serialdilutions of test antisera of 1×10⁻¹ to 1×10⁻⁸ were incubated with eachtest peptide for 30 minutes at room temperature. In some instancesantisera raised against a particular peptide of the Peptides 1-6 werepreincubated with large excesses of the other peptides of the Peptide1-6 group or with hG₁₇ or hG₃₄ in an attempt to inhibit binding of theantiserum to its particular peptide and also to demonstrate theoccurrence of antibodies in the antisera that were specific for thesequence (spacer) of each peptide that was common to all of Peptides 1-6(e.g. Arg-Pro-Pro-Pro-Pro-Cys). After washing each well thoroughly toremove unbound antibody, each well was treated with biotinylatedanti-rat immunoglobulin reagent for 30 minutes at room temperature.After another wash sequence to remove unbound anti-rat reagent,avidin-alkaline phosphatase conjugate was added and the mixture wasincubated for an additional 30 minutes. The mixture was washedthoroughly to remove unbound avidin-alkaline phosphatase reagent, andthe chromogenic substrate PNPP was added for a 10 minute period. Theabsorbance of each well was read at 490 nm after the 10 minuteincubation.

The standard RIA procedure was followed. In the RIA, 0.1, 1.0 or 10.0 ulaliquots of antiserum were incubated with approximately 200 pg of ¹²⁵ Ilabeled hG₁₇ or 400 pg of labeled hG34. The antisera were incubated withlabel for 2 hours, and were followed by a precipitation ofhormone-antibody complexes with 25% polyethylene glycol. Antigen bindingcapacities for each antiserum where then determined from the amount ofradioactive hormone precipitated. To demonstrate the specificity of thereaction of the ¹²⁵ I labeled hormone with the antisera, aliquots of theantisera were preincubated in some tests with excess amounts of thehormone that were not labeled with ¹²⁵ I to inhibit binding of theantisera to the labeled hormone.

The specificities of the antibody responses induced by Immunogens 1-6 asmeasured by ELISA are depicted in Table 1. Immunogen 1, containing thepeptide sequence of hG₁₇ (1-6), induced antibodies that reacted stronglywith hG₁₇ and hG₁₇ (1-6) peptide, but only weakly with hG₃₄ (1-6) orhG₃₄ (13-22). Antisera raised to Immunogen 1 did not react with hG₃₄.Inhibition experiments with Peptides 1-6 demonstrated that the weakreactivity of anti-Immunogen 1 antibodies with hG₃₄ (1-6) and G₃₄(13-22) peptides was due to the presence of antibodies that were inducedby the spacer sequence (-Arg-Pro-Pro-Pro-Cys) common to all the peptidesequences of Immunogens 1-6.

Immunogens 2 and 3 induced antibody responses specific for hG₁₇ thatwere much weaker than those induced by Immunogen 1 (Table 1). Inhibitionexperiments, demonstrated that the weak reactivities of anti-Imunogen 2and 3 antibodies for Peptides 1-6 are specific for the common spacersequence of Peptides 1-6.

Immunogen 4, containing the rat G₁₇ sequence, induced antibodies thatweakly reacted with Peptides 1-6, but not hG₁₇ or hG₃₄ (Table 1).Inhibition experiments demonstrated that these antibodies were directedagainst the spacer sequence common to Peptide 1-6.

Immunogen 5 hG₃₄ (1-6), induced antibodies that strongly reacted withhG₃₄ and Peptide 5, hG₃₄ (1-6), but weakly with the other peptides andnot at all with G₁₇. Inhibition experiments demonstrated that thereactivity with Peptides 1-4 and 6 was due to anti-spacer specificantibodies.

Immunogen 6, hG₃₄ (13-22), induced antibodies that reacted weakly withPeptides 1-6, but not with hG₁₇. Inhibition experiments demonstratedthat the antibodies binding Peptides 1-6 were specific for the commonspacer sequence.

                                      TABLE 1                                     __________________________________________________________________________    Reaction in ELISA with:                                                                  Peptide 1                                                                           Peptide 2                                                                           Peptide 3                                                                           Peptide 4                                                                           Peptide 5                                                                           Peptide 6                            Antisera to:                                                                          hG.sub.17                                                                        hG.sub.17 (1-6)                                                                     hG.sub.17 (1-5)                                                                     hG.sub.17 (1-4)                                                                     rG.sub.17 (1-6)                                                                     hG.sub.34 (1-6)                                                                     hG.sub.34 (13-22)                                                                    HG.sub.34                     __________________________________________________________________________    Immunogen 1                                                                           ++ +++   +     +     +     +     +      0                             hG.sub.17 (1-6)-DT     -     -     -     -                                    Immunogen 2                                                                           +  +     +     +     +     +     +      0                             hG.sub.17 (1-5)-DT                                                                       -           -     -     -     -                                    Immunogen 3                                                                           +  +     +     +     +     +     +      0                             hG.sub.17 (1-4)-DT                                                                    -  -     -           -     -     -                                    Immunogen 4                                                                           0  +     +     +     +     +     +      0                             rG.sub.17 (1-6)-DT                                                                       -     -     -           -     -                                    Immunogen 5                                                                           0  +     +     +     +     ++    +      +++                           hG.sub.34 (1-6)-DT                                                                       -     -     -     -           -                                    Immunogen 6                                                                           +  +     +     +     +     +     +      +                             hG.sub.34 (13-22)-                                                                    -  -     -     -     -     -            -                             DT                                                                            __________________________________________________________________________     +++ to +: Strongly reactive                                                   +: Weekly reactive                                                            0: No reaction                                                           

All antisera were also tested against hCCk; none of the antisera boundto hCCK.

Table 2 demonstrates the RIA-measured antigen binding capacities ("ABC")versus hG₁₇ or hG₃₄ of antisera raised against Immunogens 1-6 afterthree immunizations of rats with 0.1 mg of conjugate.

                  TABLE 2                                                         ______________________________________                                                       Mean RIA ABC (pg/ul)                                           Rats Immunized With:                                                                           hG.sub.17 hG.sub.34                                          ______________________________________                                        Immunogen 1      19.29     0.00                                               hG.sub.17 (1-6)-DT                                                            Immunogen 2      7.59      0.00                                               hG.sub.17 (1-5)-DT                                                            Immunogen 3      2.15      0.00                                               hG.sub.17 (1-4)-DT                                                            Immunogen 4      0.00      0.00                                               rG.sub.17 (1-6)-DT                                                            Immunogen 5      0.00      6.38                                               hG.sub.34 (1-6)-DT                                                            Immunogen 6      0.00      1.28                                               hG.sub.34 (13-22)                                                             ______________________________________                                    

The liquid phase RIA demonstrated that Immunogens 1-3 containing thehG₁₇ peptide sequence induced antibodies that reacted only with hG₁₇ andthat Immunogen 5 containing the hG₃₄ sequence, induced antibodies thatreacted only with G₃₄. Immunogen 6 induced very low ABC's to G₃₄.

The ELISA and RIA assays thus demonstrate the specificity of theresponses to hG₁₇ or hG₃₄ that are induced by Immunogens 1-6.

EXAMPLE 3

This example demonstrates the ability of antisera raised against Peptide1 (hG₁₇ (1-6)) to neutralize the in vivo acid stimulating activity ofhG₁₇. In this demonstration an amount of hG₁₇ is mixed with an excessamount of anti-Peptide 1 antiserum sufficient to bind to all the hG₁₇prior to injection of the complex into a normal (non-immunized) rat.

In control experiments the amount of hG₁₇ sufficient to stimulate anincrease of acid secretion of at least 100% above nonstimulated acidsecretion in normal rats was determined to be 0.4 ug of hG₁₇ hormone perkg body weight.

Antisera from the rats immunized with Immunogen 1 were pooled andstandard amounts of antisera were incubated with 200 pg ¹²⁵ I labeledhG₁₇ after incubation with increasing amounts of cold hG17 as inhibitor.Based on this inhibition study 1 ml of antiserum was capable of binding1000× the 0.4 ug/kg dose of hG₁₇ to be administered to rats. As a safetyfactor, the 0.4 ug/kg (approximately 120 ng) of hormone was mixed with2.5 ml of anti-hG₁₇ specific antiserum raised against Immunogen 1.

Rats to be injected with hG₁₇ complexed with anti-hG17 antibodies weresurgically prepared for collection of stomach secretions by the perfusedrat stomach procedure.

Under general anesthesia and following tracheostomy, the rat wascannulated via the esophagus and duodenum to allow continuous perfusionof the stomach with 0.9% saline. The stomach perfusate was collected as5 minute interval samples and was titrated for acid content byneutralization with base (sodium hydroxide). Incremental and total acidinput during the duration of the experiment and after each treatment wasdetermined.

Each control or experimental test rat was first injected with 0.4 ug/kghG₁₇ to determine the rats total acid secretory response to thistreatment. The first treatment was followed one hour later in test ratswith an injection of 0.4 ug/kg of hG₁₇ that had been premixed for onehour with 2.5 ml of anti-hG₁₇ specific antiserum. Control rats receivedan injection of hG₁₇ mixed with 2.5 ml of antiserum raised against anunrelated peptide. After one hour, a second injection of free hG₁₇ wasadministered to the test and control rats; and stomach perfusate wascollected for an additional hour. The total acid output induced by thesecond and third injections of hG₁₇ were expressed as a percentage ofthe total acid output induced by the first injection of hG₁₇.

In five rats tested by this experimental procedure there was an 81%-100%(mean=94%) reduction in the acid secreted by the perfused rat stomach inresponse to the hormone premixed with anti-hG₁₇ specific antibody(second injection) or to the third injection consisting of free hG₁₇alone. Control rats experienced little or no reduction in acid secretionstimulated by the second and third injections of hormone. FIG. 1 andFIG. 2 illustrate the responses of a control rat (FIG. 1) andexperimental rat (FIG. 2) to these treatments.

EXAMPLE 4

A major application of this invention is the active immunization ofhumans to induce specific immunity against G₁₇ for ulcer therapy andprevention. In this example, it is demonstrated that active immunizationwith an anti-G₁₇ immunogen induces antibodies that dramatically suppressG₁₇ mediated release of stomach acid.

To actively immunize rats against G₁₇, we follow the methods used toobtain antisera in the passive immunization tests as described inExample 3. An immunogen consisting of the G₁₇ (6) peptide covalentlycoupled to Diphtheria toxoid (DT) is prepared as described in Example 1.This immunogen is suspended in Phosphate Buffered Saline at aconcentration of 4.0 mg/ml. The antigen is emulsified insqualene:arlacel (4:1) vehicle, at a final ratio of 1:1(antigen:vehicle). Nor-MDP is included in the mixture to give a finalconcentration of nor-MDP of 200 ug/ml. The final concentration of theDT-G₁₇ (6) in the formulation is 2.0 mg/ml. Experimental rats areinjected with 0.25 ml of this preparation intraperitoneally. Eachinjection thus delivers approximately 500 ug of immunogen plus 50 ug ofnor-MDP. A second injection is similarly administered 21 days later.

Blood samples for antibody analysis are obtained by tail vein bleedingbefore the first injection and 14 days after each injection. Sera isprepared by allowing the blood to clot for 30 minutes at roomtemperature followed by centrifugation at 400×g to remove the clots. TheSera are stored frozen until used.

To determine the antibody responses of the immunized rats, a RIA isemployed as described in Example 2. The results of this test show thatthe immunization procedure induces high titers of antibody against G₁₇.These responses are specific for G₁₇ ; no reactivity is detected withG₃₄, with pentagastrin (the biologically active, carboxy terminalfragment of G₁₇, G₃₄, and CCK), or with CCK. The antibodies are thusdirected against the unique epitope on G₁₇ that is selectively targetedby the immunogen. These results are similar to those of Example 2.

The use of the immunogens described herein for the active immunogen isnot limited to the adjuvant, vehicle, injection schedule, etc.,described above. Any means of safely inducing immunity against G₁₇ usingthe immunogens described can be applied. This includes using alternativedosages, routes, vehicles, adjuvants, exipients, slow-release compounds,etc.

We test for the neutralization of G₁₇ 's biological activity in theimmunized animals using the perfused rat stomach method, as described inExample 3, with the important difference that we do not inject antiserainto the rats (passive immunization) because the actively immunized ratsare making their own antibodies against G₁₇. The dosages of compoundsadministered in these tests, with delivery times of 5 minutes per totaldose, are: G₁₇ =0.4 ug/kg, G₃₄ =0.8 ug/kg, and pentagastrin=2.0 ug/kg.Stomach contents sampling times are 5 minutes per sample. The stomachacid outputs are calculated as the percent of maximal acid output##EQU1## where An=the acid produced over each 5 minute sampling interval(as determined by titration with NaOH); Amax=the maximal 5 minuterelease of stomach acid upon stimulation, usually (but not necessarily)by pentagastrin; and Ab=the baseline level of acid present at the timeof a given stimulation (with G₁₇, pentagastrin, or G₃₄).

The effects of active immunization against G₁₇ upon the G₁₇ andpentagastrin ("pG") induced acid secretion are shown in FIGS. 3 and 4.The ordinate represents the percent of acid output compared to themaximal acid output induced by pentagastrin. These experiments differed,by design, in the order of G₁₇ and pentagastrin challenge. In bothcases, it is clear that in the G₁₇ immunized rats the production ofstomach acid in response to G₁₇ (FIG. 3, Peaks 2 and 3; FIG. 4, Peaks 1and 3) is substantially reduced in comparison with acid secretioninduced by pentagastrin (FIG. 3, Peak 1; FIG. 4, Peak 2). The meanreduction in the total G₁₇ mediated acid secretion in our G₁₇ immunerats is 85% (compared to pentagastrin).

We verified that the acid reductions were a direct consequence ofimmunization against G₁₇ by conducting challenges with G₁₇ orpentagastrin in control rats. The control animals were immunized in anidentical manner as the G₁₇ -immune rats, except that the controlsreceived antigen consisting of DT conjugated to an unrelated peptide(i.e., non-crossreactive with gastrin). RIAs and ELISAs, run on serafrom these animals, demonstrated that they produced high antibody titersagainst both DT and the unrelated peptide, but none against G₁₇,pentagastrin, G₃₄ or CCK. When tested for acid secretion, the controlrats responded equally well to challenges with both G₁₇ andpentagastrin. The results of such a test are shown in FIG. 5. This rulesout the remote possibility that the neutralization of G17 in the G₁₇-immune rats was caused by non-specific factors (e.g., adjuvant effects,crossreactive anti-DT antibodies, etc.).

A technical challenge presented by the perfused rat stomach assay wasthe selection of the appropriate acid stimulatory compound for use as apositive control. The exquisite specificity for G₁₇ 's unique epitopethat is characteristic of antibodies induced by our immunogen enabled usto use the ideal control compound: pentagastrin. Pentagastrin comprisesthe receptor binding/stimulatory sequence of G₁₇ and also of both G₃₄and CCK, and it is not bound by antibodies induced by our immunogen. Theresponses to pentagastrin demonstrated that our immunized animals' acidresponse mechanism to G₁₇ stimulation were functional. In addition, thepentagastrin responses established the level of acid secretion to beexpected from G₁₇ stimulation. The dosages of G₁₇ and pentagastrin,which we determined experimentally, were selected to induceapproximately equal acid secretory responses in control rats (see FIG.5). Thus, we were able to accurately quantitate reductions in acidsecretion resulting from the neutralization of G₁₇.

For completeness, we have also challenged with G₃₄. We designed ourimmunogen to specifically neutralize G₁₇ mediated acid secretion(particularly following food intake) and to have no effect upon acidoutput induced by G₃₄ (which provides for basal stomach activity). Sincethe antisera from the G₁₇ immune rats do not react with G₃₄, we expectedto see no effect upon G₃₄ 's ability to stimulate acid secretion.Indeed, as shown in FIG. 6, the immunized rats secreted normalquantities of acid in response to G₃₄ stimulation (Peaks 1 and 3). Asexpected, the injection of G₁₇ failed to induce acid secretion in theseanimals (FIG. 6, Peak 2). Both G₁₇ and G₃₄ induce strong acid secretoryresponses in control rats (immunized against an irrelevant peptide), ascan be seen in FIG. 7. Clearly, the anti-G₁₇ antibodies induced by ourimmunogen have no effect upon the functions of other molecules to whichthe antibodies do not bind. The G₁₇ (6) based immunogen described hereininduces antibodies that are specific for G₁₇ and neutralize G₁₇ 's acidreleasing activity. Such an immunogen should thus protect against andcure peptic ulcers.

EXAMPLE 5

This example demonstrated that a polymerised peptide immunogen can beconstructed and used to reduce anti-G₁₇ antibody responses. Syntheticpeptides have been produced that contain the unique epitope on G₁₇ andin addition carry reactive groups that can be selectively bound tocrosslinking agents. These peptides serve as monomers in theconstruction of a polymer immunogen. By including two or more reactivegroups in each peptide it is possible to construct multi-peptideaggregates, or polymers, by reaction of the groups with a cross-linkingagent. Such polymers are then used as immunogens to induce antibodiesagainst the G₁₇ epitope expressed by the peptide. These antibodies bindto G₁₇ in vivo and neutralize G₁₇, thus mediating an anti-ulcer effect.These polymerized peptides have an advantage in that they can be used asimmunogens by themselves without a coupling to an immunogenic carrier.

The following peptide designated as Peptide 7 was constructed: ##STR1##

The G₁₇ epitope is contained in amino acids 1-7 of the peptide. Otherepitopes eg. G₃₄ epitopes, can also be used to construct other polymerimmunogens according to the invention. Amino acids 8 and 14, which areboth Lys, contain amino groups as side groups. These amino groups act asfunctional groups which are reacted with the functional groups on thecrosslinking agent to form the crosslinked peptide polymer. Other aminoacids containing side functional groups could be substituted for Lysdepending on the reactivity of the functional group with the group onthe crosslinking agent to be used. The location of the functional aminoacids can be varied in the peptide as long as they are not positionedwithin the epitope region. Additional reactive amino acids could also beadded to increase crosslinking. These additional amino acids could bereactive with the same or alternative crosslinking agents. It followsthat more than one type of crosslinking agent can be used.

Amino acids 9-13 comprise a "spacer region" between the reactive aminoacids 8 and 14. The composition, number of amino acids and length of thespacer can be varied. If desirable, helper T-cell epitopes can also beincluded in the peptide.

Peptide 8 was synthesized and purified by standard solid phase peptidesynthesis and purification methodologies. Any other method of peptideproduction well known to those skilled in the art including recombinantDNA technology can also be used to produce the peptides of theinvention.

5.0 mg of the peptide was dissolved in 1.0 ml phosphate buffer (0.1M;pH=6.8). To this was added glutaraldehyde (Grade 1, Sigma Chemical Co.)in a 2:1 molar ratio of glutaraldehyde to peptide. The glutaraldehydewas added dropwise with stirring, at room temperature.

The reaction was allowed to proceed overnight, at room temperature, withstirring. 50.0 mgs. of sodium borohydride were then added slowly to thereaction mixture, and the mixture was stirred at room temperature for anadditional hour. The mixture was transferred to dialysis tubing, 1,000molecular weight cutoff (#132636, Spectrum Medical Industries, Inc.),and exhaustively dialyzed against saline. The peptide-polymer was storedfrozen at -20° C.

The polymer was analyzed by SDS-PAGE using a 15% polyacrylamide gel. Theelectrophoresis demonstrated that the polymerized peptide containedpolymers of various sizes comprising multiples of the peptide. Theaverage polymer contained 6 peptides, however; the size of the polymersranged up to 12 peptides per molecule.

A second polymer was made using the identical procedures, except that a20:1 molar ratio of glutaraldehyde to peptide was used. The SDS-PAGEanalysis of the second polymer similar results with respect to the sizerange as compared to the 2:1 polymer.

Each polymer preparation, 2:1 and 20:1, was used to immunize twoseparate groups of five mice per group. Prior to injecting the mice withpolymer, blood samples were taken from each mouse. The preparation wassuspended in Freunds Complete Adjuvant ("FCA") H37Ra (DIFCO Labs) in a1:1 (vol:vol) ratio of polymer:FCA. The mice were each injectedintraperitoneally with 100 ug polymer in 0.2 ml of the mixture. After 21days each mouse was given a second injection of the same polymer withwhich it had been injected previously. In the second injection, theantigen was administered intraperitoneally in saline, at 100 ug permouse. Each mouse was bled 14 days after the second injection and thesera were isolated. The mouse sera were assayed for anti-G₁₇ antibodiesby radioimmunoassay (RIA). 1.0 ul of sera was added to 300 ul of buffer(1% BSA in phosphate buffered saline with 0.005M EDTA, pH=7.2). To eachof these samples was added 100 ul or 3000 CPM of ¹²⁵ I-labeled G₁₇ (NEN,Specific activity=12 uCi/ug). The samples were incubated 1.0 hour atroom temperature. We next added 100 ul of Calf Serum (Hyclone Labs),immediately followed by 500 ul of 25% polyethylene glycol-8000 (Sigma).The samples were mixed and then centrifuged for 30 minutes at 500×g atroom temperature. The supernatant was discarded, and the pelletsuspended in 250 ul of saline at 90° C. The suspension was transferredto 3.0 ml of Scintiverse II [Fisher Scientific] in mini vials for liquidscintillation counting. The samples were counted in a Beckman LiquidScintillation counter (#LS 5000 LE) for ¹²⁵ I. The binding capacities ofthe antisera were calculated from the resulting ¹²⁵ I counts per sampleand are depicted in FIG. 8.

Both of the polymers induced anti-G₁₇ antibody responses. Polymer #1,the 2:1 ratio polymer (FIG. 8), induced a very strong response of 56 pgof antigen bound per ul of sera. Polymer #2, the 20:1 ratio polymer(FIG. 8), induced a response that was 10-fold lower. The responseinduced by polymer #1 is equivalent to that induced by three injectionsof the G₁₇ (6)-DT immunogen of Example 2 in rats.

Thus, polymerized synthetic peptides can be used to induce potentanti-G₁₇ antibody responses.

EXAMPLE 6

The following example demonstrates that anti-G₁₇ antibodies neturalizethe tumor stimulatory activity of G₁₇ in vitro.

HCT-116 cells (a human colon cancer cell line) were cultured in McCoy's5a medium (McCoy et al., Proc. Soc. Exper. Biol. Med. 100:115-118)supplemented with epidermal growth factor (10 ng/ml), insulin (20ug/ml), transferrin (4 ug/ul), sodium selenite (10⁻⁸ M), hydrocortisone(2 ug/ml), and triiodothyronine (4×10⁻¹⁰ M). Subcultures were made onceweekly for four weeks by treating cultures with 0.5% Trypsin+0.2% EDTAin Hanks Balanced Salts Solution to remove adherent cells, followed byinoculation of T-75 tissue culture flasks with approximately 1×10⁶cells. Cultures were maintained under standard conditions (37°, 100%humidity, 5% CO₂).

Prior to testing, the HCT-116 cells were synchronized to late G₀ phasewith thymidine, as follows: the HCT-116 cells were seeded into 24 wellculture plates at approximately 1×10⁴ cells per well and incubatedovernight in 1 ml supplemented McCoy's medium 5a. The medium was thenremoved and replaced with fresh supplemented medium. Thymidine was addedto 0.8 mM final concentration to each well and the cultures wereincubated for 24 hours. At the end of the synchronization period, themedium containing thymidine was replaced with test media as describedbelow.

To demonstrate that HCT-116 cells proliferate under the influence ofgastrin, the synchronized HCT-116 cells were grown in supplementedMcCoy's 5a medium, in the presence or absence of 10 uM pentagastrin (thehormonally active segment of gastrin). To assess cell proliferation,total cell counts were performed after selected incubation times. Asshown in FIG. 10, HCT-116 cells proliferated more rapidly in thepresence of pentagastrin than in the absence of the hormone. Thisdifference was already evident after three days of culture, and wasmaximal by day five. The increase in the number of cells of pentagastrintreated culture at the end of eight days was three times greater thanthat of the non-pentagastrin treated cultures.

To demonstrate that anti-G₁₇ antibodies neutralize the proliferativeactivity of G₁₇, the effect of anti-G₁₇ antisera upon G₁₇ -inducedincreases in the rate of [³ H]-thymidine uptake by HCT-116 cells wasstudied.

Synchronized HCT-116 were cultured in 24 well plates of 10⁴ cells/wellin supplemented McCoy's 5a medium without FBS. The total culture volumewas 1 ml per well. Four wells were cultured for each test condition.Cells were grown in presence of G₁₇ at two concentrations, 5 uM and 50uM. At each G₁₇ concentration, rat anti-human G₁₇ antisera (antigenbinding capacity determined by RIA=30 pg/ul) or normal rat sera wereadded at a final dilution of 1:25.

After 8 days of culture, 0.4 uCi of [³ H] thymidine (specific activity=2Ci/mMole) were added to each test well. Following 16 hours incubation,the replicates were processed with a Multiple Automated Cell Harvester(Mini-Mash II, Whittaker Bioproducts) and the [³ H]-thymidineincorporation was determined by scintillation counting.

As shown in Table 3, the addition of anti-G₁₇ antiserum caused asubstantial reduction in [³ H]-thymidine incorporation, relative touptake by cells cultured in the presence of normal rat serum. At 5 uMG₁₇, counts incorporated were reduced by 59%; at 50 uM G₁₇, label uptakewas reduced by 34%. As [³ H]-thymidine incorporation directly reflectscell proliferation, this test shows that anti-G₁₇ antiserum inhibits theproliferation activity of human G₁₇ on colon cancer cells.

                  TABLE 3                                                         ______________________________________                                                      Mean CPM [.sup.3 H] Thymidine                                   Quantity of Human G.sub.17                                                                  Incorporated (+/- SE)                                           in Culture    Normal Rat Serum                                                                            Anti-hG.sub.17 Serum                              ______________________________________                                        50 uM         2130 ± 141 1404 ± 310                                      5 uM         1621 ± 131 664 ± 206                                      ______________________________________                                    

EXAMPLE 7

The following experiment was performed to demonstrate that the growth ofestablished HCT-116 tumors is retarded when nude mice bearing the tumorsare treated with anti-gastrin immunoglobin.

Anti-human G₁₇ serum was obtained from rats immunized against hG₁₇(6)-DT (i.e., Immunogen 1). Normal serum was obtained from nonimmuizedrats. Immunoglobulin fractions of the anti-G₁₇ serum and the normal ratserum were prepared by affinity chromatography, using Protein ASepharose. Both immunoglobulin preparations were adjusted to a finalprotein concentration of 1 mg/ml in PBS. Measured by RIA, the G₁₇-direct antigen binding capacity (ABC) of the anti-human G₁₇immunoglobulin preparation was 30 pg/ul. The normal rat immunoglobulinpreparation had no anti-human G₁₇ activity.

Thirty nude mice were each implanted subcutaneously (dorsally near theleft shoulder) by trocar needle (14 gauge) with a single 2 mm cube ofHCT-116 tumor tissue. The tumors were allowed to grow for one week priorto random assignment of individual mice to one of three treatmentgroups, ten mice per group, on day 0 of the Test.

Group I was treated with anti-human G₁₇ immunoglobulin function. Eachmouse in Group I received intraperitoneal injections of 0.5 mg. ofimmunoglobulin beginning on day 0 and repeated on days 4, 8, and 14 ofthe test. The Mean human G₁₇ ABC of the mouse sera on day 16 was 8.3±1.3pg/ul.

Group II was treated with normal rat immuglobulin fraction. Each mousereceived intraperitonael injections of 0.5 mg of immunoglobulinbeginning on day 0 and repeated on days 4, 9, and 14, of the test. Onday 16, the human gastrum G₁₇ ABC's of those sera was opg/ul.

Group III received no immunoglobulin injection but was injected insteadwith saline on days 0, 4, 9, and 14 of the test. The human gastrin G₁₇ABC's of those sera was Opg./ul. on day 16.

On day 1, mice of Groups I and II were implanted subcutaneously withosmotic pumps (Alzet 2002) that were charged with human G₁₇ thatdelivered 10 ug/day human G₁₇ continuously for 14 days. On day 1, miceof Group III were implanted subcutaneously with osmotic pumps chargedwith saline which delivered a dose of saline instead of hormonecontinuously for 14 days.

Each mouse was observed daily for changes in the size of theirindividual tumors. Measurements of the tumors were made by verniercaliper approximately every other day. Volumes of tumors were estimatedby the calculation: volume=(length×width²)÷2 (Euhus et. al., 1986, J.Surg. Oncol. 31:229-234).

Observations on Group II mice were made through day 17 of the test, onwhich day the Group II mice were euthanized. Observations on Groups Iand II were continued until day 32 of the test.

As shown in FIG. 11, tumors in Group II mice (administered human G₁₇ andtreated with normal rat immunoglobulin) grew very rapidly in response tothe added gastrin, increasing more than 120 fold in volume in 16 days.

Tumors of Group I mice (administrated human G₁₇ and treated withanti-human G₁₇ immunoglobulin) grew at a significantly slower rate thanthe tumors of Group II. By day 16, the volume of the tumors of Group Imice were, on average, approximately 11 times smaller than those ofGroup II mice (Table 4). The results of this test demonstrate thatHCT-116 tumors are stimulated to grow by human gastrin G₁₇ and thattreatment with anti-human G₁₇ specific immunoglobulin neutralizes thisgrowth-promoting effect and significantly slows the growth of HCT-116tumors.

Tumors of Group III mice (not administered hG₁₇ and no immunoglobulintreatment) grew at a faster rate than Group I (FIG. 12), suggesting anautocrine production of human G₁₇ (by the HCT-116 tumor cells) thatstimulated the tumors to grow. Tumor-produced G₁₇ would be neutralizedin the Group I animals, due to the injected anti-G₁₇ immunoglobulins. Onday 32 of the test, the Group III tumors had attained approximately thesame volume attained by Group II tumors on day 16. However, on day 32tumors of Group I were significantly smaller than tumors of Group III(i.e., 3.3 times smaller volume, Table 4), indicating an inhibitoryeffect of the anti-G₁₇ immunoglobin on tumor growth.

                  TABLE 4                                                         ______________________________________                                                           Mean Tumor Volume                                                             (mm3 ± SE)                                              Group Treatment          day 16    day 32                                     ______________________________________                                        I     Anti-G.sub.17 Antibody plus G.sub.17                                                             142 ± 38                                                                              545 ± 137                              II    Normal Rat Serum plus G.sub.17                                                                   1512 ± 348                                                                           --                                         III   Saline and no G.sub.17                                                                           391 ± 63                                                                             1825 ± 313                              ______________________________________                                    

EXAMPLE 8

The following test demonstrates that antibodies against human G₁₇inhibit tumor development and growth in nude mice that have beeninjected with suspended cells of the human colon cancer line, HCT-116.

Anti-human G₁₇ serum was obtained from rats immunized against hG₁₇(6)-DT. Normal rat serum was obtained from nonimmunized rats.Immunoglobulin fractions of the anti-G₁₇ serum and the normal rat serumwere prepared by affinity chromatography, using Protein A Sepharose.Both immunoglobulin preparations were adjusted to a final proteinconcentration of 1 mg/ml in PBS. Measured by RIA, the G₁₇ -directantigen binding capacity (ABC) of the anti-human G₁₇ immunoglobulinpreparation was 30 pg/ul. The normal rat immunglobulin preparation hadno anti-human G₁₇ activity.

HCT-116 cells were grown in vitro in the presence of pentagastrin asdescribed in Example 6. Cells in the log phase of growth were collectedfrom in vitro culture, washed by centrifugation in PBS, and resuspendedto 10 cells/ml. Viability was assessed by trypan blue exclusion.

Each of 20 nude mice (NIH strain) were injected subcutaneously on theirdorsal side near the right shoulder with a single bolus of 5×10⁶ cells.

Two days after the injection of HCT-116 cells, the mice were randomlyassigned to two groups of ten mice each. One group (Treated Group) wasinjected intraperitoneally with 0.5 mg per mouse of the anti-human G₁₇immunoglobulin preparation. The other group (Control Group) was treatedwith 0.5 mg per mouse of the normal immunoglobulin fraction. Seven dayslater these treatments were repeated. These treatments resulted in seraG₁₇ binding capacities of 11.1 pg/ul in the anti-G₁₇ immunoglobulintreated mice and 0 in the normal immunoglobulin treated mice (see Table5).

Two days after injections of the HCT-116 cells, all of the mice werestarted on daily injections of human G₁₇ for 16 consecutive days. Atotal daily dose of 51 ug of hormone per mouse was administered in 3separate injections of 17 ug each, given at 4 hour intervals. The micewere bled on day 16 of the test to determine binding capacities by RIA.

Daily observations on the occurrence and growth of tumors were madevisually and by palpation. On day 18 of the test, the tumors weremeasured by vernier caliper and the volume of each tumor estimated bythe following formula: volume=(length×width²)÷2 (Euhus et. al., 1986, J.Surg. Oncol. 31:229-234). On Day 18, the mice were euthanized and thosemice without visually detectable tumors were dissected and furtherexamined for tumors under a stereo microscope at 10× magnification.

As shown in Table 5, the anti-human G₁₇ immunoglobulin prevented tumorsfrom developing in six out of ten mice in the Treated Group. Only onenormal immunoglobulin-treated mouse failed to develop a tumor over thecourse of the test. In the four anti-G₁₇ treated animals that developedtumors, the mean tumor volume was reduced greater than four-foldcompared to the tumors that developed in the mice treated with normalrat immunglobulin. The Results demonstrate that treatment withanti-human G₁₇ immunoglobulin inhibits the development and growth ofHCT-116 tumors in nude mice.

                  TABLE 5                                                         ______________________________________                                                     Treatment                                                                     Normal Rat Anti-G.sub.17                                                      Immunoglobulin                                                                           Immunoglobulin                                        ______________________________________                                        Mean Sera Anti-G.sub.17 ABC                                                                  0.0 pg/ul    11.1 pg/ul                                        Titer (Range)               (7.6-14.8)                                        Number of Mice Develop-                                                                      9            4                                                 ing Tumors                                                                    Mean Tumor Volume ±                                                                       21.2 ± 11.2 mm                                                                          4.7 ± 2.8 mm                                   S.E.                                                                          ______________________________________                                    

EXAMPLE 9

This test demonstrates that antibody-mediated immunity to G₁₇ can beselectively and safely reversed by the injection of peptide capable ofbinding to the antibody.

Six rats that were twice previously immunized with human gastrin G₁₇(6)-DT immunogen and which exhibited anti-human gastrin antigen bindingcapacity of 17-34 pg/ul of serum were prepared for the standard stomachperfusion procedure. To demonstrate that each immunized rat was able toinhibit the acid secretion stimulatory activity of human G₁₇, a standarddose of approximately 120 ng of G₁₇ hormone was administered to eachrat. After measuring the human gastrin G₁₇ stimulated acid output inresponse to the 120 ng of G₁₇, a second dose of human gastrin G₁₇, of2.5 ug of hormone at twenty times the first dose was given to each ratand the gastrin stimulated acid output was measured again.

Two of the rats were then euthanized and their kidneys removed forsectioning and examination for deposition of complexes of gastrin andanti-gastrin antibody. Two of the remaining rats were then euthanizedand their kidneys removed for sectioning. The remaining two rats weregiven 250 ug of human gastrin G₁₇, were followed for gastrin stimulatedacid secretion, and then were euthanized and their kidneys were removedfor sectioning.

Rapid reversal of human gastrin G₁₇ neutralizing activity of theanti-sera of human gastrin G₁₇ immunized rats is demonstrated in FIG. 9.As expected from our previous perfusion assays, administration ofapproximately 120 ng of human gastrin G₁₇ resulted in an 87% meaninhibition of the expected gastrin stimulated acid secretion of all therats. Challenge with 2.5 ug of human gastrin G₁₇ resulted in a gastrinstimulated acid response identical to that seen in non-immunized rats.Challenge with 25 ug or 250 ug of human gastrin G₁₇ resulted in anexaggerated acid secretion response. Kidney sections taken from theserats treated with large amounts of human gastrin G₁₇ were all negativefor formation and deposition of immune complexes.

Treatment of rats immunized with human gastrin G₁₇ with only 2.5 ug ofhuman gastrin G₁₇ immediately reversed the neutralization of gastrinstimulated acid secretion that was observed in these rats when they werefirst challenged with 120 ng of human gastrin G₁₇. Based on the antigenbinding capacities of these rats, which exhibited a range of 17-34 pg/uland a mean of 24 pg/ul, 2.5 ug of G₁₇ is at least a four-fold excess ofhormone injected over the total antigen binding capacity of the rat'sserum. Such a small amount of hormone, if given in the same proportionbased on body weight to humans would amount to a range of only 500-700ug of human G₁₇.

Preferably the antibody neutralizing by infusion would utilize amonovalent neutralizing molecule that bears the gastrin epitope butwhich does not itself induce acid secretion (e.g., by changing theC-terminal end of G₁₇).

The antibody neutralizer will not prevent a renewed production ofanti-gastrin antibodies, the duration of which is determined by theconditions of immunization. However, it will neutralize the antibodiesas they are produced. In practice, it may be necessary in most cases toprovide for neutralization of antibodies synthesized after the initialdose of neutralizing compound is administered. This could beaccomplished by means of additional infusions or, preferably, throughthe administration of the neutralizer in a sustained release compound ordevice. Although such administration would continue until the synthesisof anti-gastrin antibodies ceases, the quantity of antibody to beneutralized would be significantly less than that eliminated by thefirst administration of neutralizer. Consequently, the dose/frequency ofneutralizer adminstrations would be diminished as antibody productionsubsides.

This invention and its preferred embodiments have been described indetail. It will be appreciated that those skilled in the art, uponconsideration of this disclosure, may make modifications andimprovements within the scope of this invention.

We claim:
 1. An immunogen which induces a sufficient level of antibodiesin an immunized animal to neutralize the physiological effect of thepeptide hormone G₁₇ in the immunized animal, comprising an immunogeniccarrier conjugated by a spacer peptide sequence to the carboxy terminusof a peptide containing a single antigenic epitope cross-reactive withan epitope on the amino terminus of G₁₇, which immunogen inducesantibodies in the immunized animal which react with an epitope on G₁₇which is present on the first six amino acid residues beginning from theamino terminus of G₁₇ and do not cross-react with any other epitope onG₁₇ or the peptide hormone G₃₄ and prevent the binding of the G₁₇ to itsphysiological receptors in the immunized animal.
 2. An immunogenaccording to claim 1 wherein the carboxy terminus of the peptide iscoupled to the immunogenic carrier by a spacer peptide sequence.
 3. Animmunogen according to claim 1 wherein the peptide is selected from thegroup consisting of pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu, andpGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu.
 4. An immunogen according to claim2, or 3, wherein the immunogenic carrier is selected from the groupconsisting of diphtheria toxoid, tetanus toxoid, keyhole limpethemocyanin, and bovine serum albumin.
 5. The immunogen of claims 1, 2,or 3, wherein the immunized animal is a mammal.
 6. The immunogen ofclaim 5, wherein the immunized animal is a human.
 7. The immunogencomprising the peptide having the sequencepGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu, conjugated by its carboxylterminal to a spacer peptide sequence which spacer sequence isconjugated to an immunogenic carrier.
 8. A pharmaceutical compositioncomprising an immunogen which comprises an immunogenic carrierconjugated by a spacer peptide sequence to the carboxy terminus of apeptide which contains a single epitope which is cross-reactive with theepitope present on the first six amino acids of the N-terminal aminoacid sequence of heptadecagastrin ("G₁₇ ") and a pharmaceuticallyacceptable carrier or adjuvant.
 9. A pharmaceutical compositionaccording to claim 8, wherein the peptide comprises a fragment of theN-terminal amino sequence of G₁₇, which is not contained in theN-terminal amino acid sequence of G₃₄.
 10. A pharmaceutical compositionaccording to claim 8, wherein the peptide is selected from the groupconsisting of pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu,pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu, pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu,pGlu-Gly-Pro-Trp-Leu-Glu-Glu, pGlu-Gly-Pro-Trp-Leu-Glu andpGlu-Gly-Pro-Trp-Leu.
 11. A pharmaceutical composition according toclaim 8, wherein the immunogenic carrier is selected from the groupconsisting of diphtheria toxoid, Tetanus Toxoid, keyhole limpethemocyanin, and bovine serum albumin.
 12. A peptide selected from thegroup consisting of, pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu,pGlu-Gly-Pro-Trp-Leu-Glu-Glu, pGlu-Gly-Pro-Trp-Leu-Glu,pGlu-Gly-Pro-Trp-Leu, and pGlu-Gly-Pro-Trp.
 13. A pharmaceuticalcomposition comprising of the peptide of claim 12 conjugated by a spacerpeptide sequence attached to its carboxy terminal end to an immunogeniccarrier.